Growth differentiation factor promoter and uses therefor

ABSTRACT

GDF promoters (e.g., GDF-8 promoters) are described. Also described are methods of using the GDF promoters to regulate tissue-specific gene expression, and to identify compounds which regulate GDF expression.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/632,879, filed on Aug. 4, 2000, which is a divisional of U.S.application Ser. No. 09/354,409, filed on Jul. 15, 1999, which claimspriority to U.S. provisional application No. 60/092,865, filed on Jul.15, 1998, and U.S. provisional application No. 60/123,270, filed on Mar.8, 1999, incorporated herein in their entirety by this reference.

FIELD OF THE INVENTION

The invention relates to GDF promoters, such as GDF-8 promoters, as wellas methods of using them, e.g., methods for screening for regulatorycompounds of GDF-8 expression.

BACKGROUND OF THE INVENTION

GDF-8 is a member of the TGF-β superfamily, which encompasses a largegroup of growth and differentiation factors that play important roles inregulating embryonic development and in maintaining tissue homeostasisin adult mammals. GDF-8 appears to function specifically as a negativeregulator of skeletal muscle growth and, therefore, has potentialapplications in producing livestock and game animals, such as cows,sheep, pigs, chicken, turkey, and fish which are relatively high inmusculature and protein, and low in fat content. In addition, GDF-8 haspotential applications in various cell proliferative and differentiationdisorders, especially those involving muscle, nerve and adipose tissuesin both human and animals. GDF-8 also appears to be involved in glucosetransport and, therefore, has potential applications in the treatment ordiagnosis of glucose transport associated disorders such as diabetes.

Many drug and diet regimens exist which may help increase muscle andprotein content and lower undesirably high fat and/or cholesterollevels, but such treatment is generally administered after the fact, andis begun only after significant damage has occurred to the vasculature.Accordingly, it would be desirable to produce animals which aregenetically predisposed to having higher muscle content, without anyancillary increase in fat levels. U.S. patent application Ser. No.09/019,070, inventors Se-Jin Lee and Alexandra C. McPherron, filed Feb.5, 1998 and entitled Growth Differentiation Factor-8 also describes theproduction of GDF-8, as well as potential uses. This application is alsohereby incorporated by reference.

Control of GDF-8 gene expression is highly desirable. The availabilityof discreet DNA segments which are capable of conferring either anegative or positive control capability to known genes in eukaryoticsystems is generally lacking in the art. Isolation of regulatory geneticsequences for GDF-8 is disclosed in the present invention.

SUMMARY OF THE INVENTION

The present invention relates to the molecular regulation of GDF-8expression and, in particular, to the isolation and identification ofregulatory sequences of GDF gene promoters, such as the GDF-8 genepromoter.

In one embodiment, the present invention provides the completenucleotide sequence and identification of genetic regulatory elementswhich promote expression of GDF-8.

In another embodiment, the present invention provides a method ofscreening for compounds which regulate GDF-8 expression, for example, byinhibiting or by stimulating GDF-8 expression.

In yet another embodiment, the present invention provides a DNAexpression construct containing the GDF-8 promoter operatively linked toa gene of interest (GOI), and a method of expressing a GOI in muscle andother tissues (e.g., tissues in which GDF-8 is naturally expressed).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show the results of a human GDF-8 promoter-luciferasereporter construct transfection assay in 6 different cell lines.

FIG. 2 shows the nucleotide sequence corresponding to the human GDF-8promoter element (SEQ ID NO:1). The initiation codon, ATG, is underlinedin bold letters. The boxed sequences are the three mutated regions. Thenumbers represent the nucleic acid positions upstream of the ATG.

FIG. 3A is a schematic representation of various human GDF-8 promoterreporter constructs. FIG. 3B is a graph showing the Luciferase activityof these constructs in RD (human embryonal rhabdomyosarcoma) cells.Luciferase activities are expressed as percent of control (pGL3-0.65,luciferase reporter plasmid containing 0.65 Kb of sequence upstream ofthe ATG site). The relative luciferase activities of the reporterplasmids are normalized to the β-galactosidase activity.

FIG. 4A is a schematic representation of various mutated human GDF-8promoter-luciferase reporter constructs. FIG. 4B is a graph showing theluciferase activity of these constructs in RD cells. Luciferaseactivities are expressed as percent of control (pGL3-0.65, luciferasereporter plasmid containing 0.65 Kb of sequence upstream of the ATGsite). The relative luciferase activities of the reporter plasmids arenormalized to the β-galactosidase activity.

FIG. 5A shows the double stranded DNA oligonucleotide sequences used inconstructing human GDF-8 promoter-luciferase reporter constructscontaining concatemers of CAAATG and GACAGC sequences. Box 1 sequencecorresponds to SEQ ID NO: 2. Box 3 sequence corresponds to SEQ ID NO: 3.FIG. 5B is a graph showing the luciferase activity of these constructsin RD cells. Luciferase activities are expressed as percent of control(pGL3-0.65, luciferase reporter plasmid containing 0.65 Kb of sequenceupstream of the ATG site). The reporter construct pGL-0.21 is theparental plasmid of the concatemer reporter constructs. The relativeluciferase activities of the reporter plasmids were normalized to theβ-galactosidase activity.

FIG. 6 is a graph showing the luciferase activities of the variousstable RD clones containing the pGL3-0.65 luciferase reporter sequencereferred to in the description of FIG. 5B, and the effects of TGF-β andTNFα on luciferase reporter expression.

FIG. 7A shows the nucleotide sequence for the mouse GDF-8 promoterregion (SEQ ID NO: 4). FIG. 7B shows the nucleotide sequence for thechicken GDF-8 promoter region (SEQ ID NO: 5).

FIG. 8 shows an alignment of the nucleotide sequences for human, mouse,pig and chicken GDF-8 promoter elements (SEQ ID NOS: 6, 7, 8 and 9,respectively) upstream of the TATAA box (underlined). The CAAATG, CAGACAand GACAGC sequences are boxed. The shaded areas represent regions ofsequence homology.

DETAILED DESCRIPTION OF THE INVENTION

The term a “GDF promoter,” as used herein, refers to nucleotide sequenceelements located upstream of the 5′ end of a GDF gene (e.g., a geneencoding GDF-8 or another related or another homologous growth factor)which regulate (e.g., initiate, upregulate or downregulate)transcription and/or expression of the gene. For example, a GDF promotercan include sequence elements necessary to initiate gene transcription,enhancer elements, repressor elements and other cis-acting controlelements which modulate gene expression. Accordigly, the term “GDFpromoter” is used herein interchangeably with the term “GDF regulatoryregion” or “GDF control region.”

An “isolated GDF promoter” refers to a GDF promoter which is removedfrom its natural sequence context. For example, an isolated GDF promotercan be a GDF promoter cloned or otherwise removed from its naturalsource, and inserted upstream from the 5′ end of a heterologousstructural gene, e.g., within an expression vector. The term “GDFpromoter” also refers to nucleotide sequences having sufficient homologyto a GDF promoter that it exhibits one or more functions of the GDFpromoter (e.g., drives transcription of a gene operably linked to thepromoter). Generally, GDF promoters are derived from the 5′ flankingregion of a GDF gene.

GDF promoters of the invention include those derived from any GDF gene,such as the GDF-8 gene or a homologous gene (e.g., GDF-11). The term“derived from”, as it is used herein, refers to a source or origin foran isolated GDF promoter of the invention. For example, a GDF promoterthat is “derived from” a particular GDF gene (e.g., a GDF-8 gene) willbe identical or highly homologous in nucleotide sequence to the GDFpromoter of a naturally occurring GDF gene (e.g., a GDF-8 gene).Isolated GDF promoters of the invention which are “derived from” GDFgenes, such as GDF-8 genes, also include those which have been modifiedby insertion, deletion or substitution of one or more nucleotides butwhich retain substantially the same activity or function.

The term “a GDF gene” refers to a GDF gene from any naturally possessingthe GDF gene, including, but not limited to human, chicken, cow, sheep,fish, pig and mouse. A “GDF gene” also refers to a GDF gene from anypiscine, crustacean or mollusk naturally possessing the GDF gene. In aspecific embodiment, the invention provides a human GDF-8 promotercomprising all or a portion (or portions) of the nucleotide sequenceshown in FIG. 2 (SEQ ID NO:1), as well as GDF-8 promoters from othermammals having regions of homology to the human promoter sequence,particularly in regions required for activity of the promoter sequence.Generally, this range of homology is about 60% to 90% or higher. Forexample, homologous regions within GDF-8 promoters from human (SEQ IDNO:6), mouse (SEQ ID NO:7), pig (SEQ ID NO:8) and chicken (SEQ ID NO:9)GDF-8 genes are shown in FIG. 8.

Accordingly, GDF promoters of the invention include promoters having anucleotide sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,98%, or more identical to the nucleotide sequence set forth in SEQ IDNos: 1, 4, 5, 6, 7, 8 and 9, and which modulate expression of a geneoperably linked to the promoter. To determine the percent identity oftwo nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, or 90% of the length of the referencesequence. The nucleotides at corresponding nucleotide positions are thencompared. When a position in the first sequence is occupied by the samenucleotide as the corresponding position in the second sequence, thenthe molecules are identical at that position (as used herein nucleicacid “identity” is equivalent to nucleic acid “homology”). The percentidentity between the two sequences is a function of the number ofidentical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twonucleotide sequences is determined using the GAP program in the GCGsoftware package (available at http://www.gcg.com), using aNWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and alength weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percentidentity between two nucleotide sequences is determined using thealgorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which hasbeen incorporated into the ALIGN program (version 2.0), using a PAM120weight residue table, a gap length penalty of 12 and a gap penalty of 4.

Accordingly, GDF promoters of the invention can be identified bycomparing the regions 5′ of the transcription initiation site of GDFgenes to the specific GDF promoter sequences provided herein (e.g., SEQID NO: 1 corresponding to the human GDF-8 promoter) and looking forregions of homology, corresponding to the active promoter sequences. Thespecific GDF-8 promoter sequences provided by the present invention alsocan be used to screen for homologous sequences from other species usingstandard DNA hybridization protocols (e.g., under conditions of highstringency).

GDF promoters of the invention contain DNA sequence elements whichensure proper binding and activation of RNA polymerase, influence wheretranscription will start, and affect the level of transcription. Inaddition, specific regulatory sequences that are functional in theregulation (induction and repression) of gene expression responsive tostimuli or specific chemical species also may be included within thepromoter sequence.

A DNA “coding sequence”, “coding region”, or a “sequence encoding” aparticular protein is a DNA sequence which is transcribed and translatedinto a polypeptide in vitro or in vivo when placed under the control ofappropriate regulatory elements. The boundaries of the coding sequenceare determined by a start codon at the 5′-terminus and a translationstop codon at the 3′-terminus. A coding sequence can include, but is notlimited to, cDNA from eukaryotic mRNA, genomic DNA sequences fromeukaryotic (e.g., mammalian, animal, avian etc.) sources, and evensynthetic DNA sequences. A transcription termination sequence willusually be located 3′ to the coding sequence.

The term “reporter gene”, as used herein, refers to a gene encoding aprotein which is readily quantifiable or observable. Because generegulation usually occurs at the level of transcription, transcriptionalregulation and promoter activity are often assayed by quantitation ofgene products. For example, promoter regulation and activity has oftenbeen quantitatively studied by the fusion of the easily assayable E.coli lacZ gene to heterologous promoters (Casadaban and Cohen (1980) J.Mol. Biol. 138:179-207). The structural gene for chloramphenicol acetyltransferase (CAT), green fluorescence protein (GCFP), and luciferase areother genes commonly used to detect activity of a promoter or otherregulatory sequence.

The term “tissue-specific expression”, as it is used herein, refers to alimited or characteristic pattern of gene expression among cell types.In other words, expression of a gene is observed in certain tissues ofan organism but not in other tissues. For example, “muscle-specific”expression of a gene denotes that that gene is expressed in the muscleand perhaps limited other tissues, but is not expressed in all tissues(e.g., globally).

A “vector” is a replicon, such as a plasmid, phage, or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment. An “expression vector” means any DNA vector(e.g., a plasmid vector) containing the necessary genetic elements forexpression of a desired gene, including a promoter region of the presentinvention. These elements are “operably linked” to the gene, meaningthat they are located at a position within the vector which enables themto have a functional effect on transcription of the gene. The regulatoryelements need not, be contiguous with the coding sequence, so long asthey function to direct the expression thereof. Thus, for example,intervening untranslated yet transcribed sequences can be presentbetween a promoter and the coding sequence and the promoter can still beconsidered “operably linked” or “in operable linkage to” the codingsequence.

A cell has been “transformed” by exogenous DNA (e.g., a transgene) whensuch exogenous DNA has been introduced inside the cell membrane.Exogenous DNA may or may not be integrated into the chromosomal DNAcomprising the genome of the cell. With respect to eukaryotic cells,though, a stably transformed cell is one in which the exogenous DNA hasbecome integrated into the chromosome such that it is inherited bydaughter cells though chromosome replication.

A “host cell” is a cell that has been transformed, or is capable oftransformation, by an exogenous nucleic acid molecule.

A “transgene” refers to a nucleic acid which is introduced into a cell.Typically, the transgene is integrated into the genome of the cellfollowing introduction. The transgene can encode a protein which is notexpressed in the cell or which is expressed in the cell at low levels orin defective form.

A “transgenic animal” is an animal carrying in its cells at least onetransgene. For example, the transgenic animal can contain in its cells atransgene corresponding to a gene of another species which has beenintroduced into the germline of the animal, such that the introducedgene is present in all somatic and germline cells.

GDF promoters of the invention can vary in size. Generally, the promotersequence spans or is located within approximately 500 bases to 3000bases of sequence in the 5′ direction (or upstream) to the site oftranscription initiation. However, the promoter can include sequencesout to approximately 4000 bp or further 5′ to the site of transcriptioninitiation. When employed in the context of a heterologous structuralgene, the optimal location of the GDF promoter with respect to thetranscription initiation site can vary. Generally, the same benefit willbe obtained when the GDF promoter is located anywhere up to about 300nucleotides or more upstream from the transcription initiation site.However, in a preferred embodiment, the GDF promoter is located within150 nucleotides of the transcription initiation site.

The majority of promoters control initiation of transcription in onedirection only. Therefore, in order to be under the control of a GDFpromoter of the invention, a structural gene generally must be locateddownstream (in the 3′ direction) of the GDF promoter and in the correctorientation with respect to the promoter. One or several genes may beunder the control of a single GDF promoter or, conversely, one or moreGDF promoters may control a single structural gene.

In one embodiment, the GDF promoter regulates expression of a geneoperatively linked to the promoter by changing the ability of RNApolymerase to bind to DNA sequences within the GDF promoter. Forexample, a regulatory protein can bind to a DNA sequence at or near theposition of RNA polymerase binding to enhance or prevent transcription.Alternatively, a regulatory protein (e.g., an inducer or repressormolecule) can directly or indirectly interact with RNA polymerase itselfto change its specificity for recognition and binding to a DNA sequenceof the GDF promoter. In either case, specific sequence(s) within the GDFpromoter are involved in the mechanism of regulation.

The term “recombinant DNA molecule” is used herein to distinguish DNAmolecules in which heterologous DNA sequences have been artificiallycleaved from their natural source or ligated together by the techniquesof genetic engineering, for example, by in vitro use of restrictionenzymes or ligation using DNA ligase.

GDF promoters of the invention can be identified and cloned from theirnatural sources (e.g., the genome of a human, mouse, chicken, pig, cow,fish or sheep). The process of cloning a DNA fragment involves excisionand isolation of the DNA fragment from its natural source, insertion ofthe DNA fragment into a recombinant vector and incorporation of thevector into a microorganism or cell where the vector and inserted DNAfragment are replicated during proliferation of the microorganism orcell. The term “cloned DNA fragment” or “cloned DNA molecule” refers toa DNA fragment or molecule produced by the process of cloning, as wellas copies (or clones) of the DNA fragment or molecule replicatedtherefrom. Standard techniques for cloning, DNA isolation, DNAamplification and purification, enzymatic reactions (e.g., involving DNAligase, polymerase, or restriction endonucleases) and various separationtechniques, which known and commonly employed by those skilled in theart, can be used in the present invention. A number of these standardtechniques are described in: Maniatis et al. (1982) Molecular Cloning,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Wu (ed.)(1979)Meth. Enzymol 68; Wu et al. (Eds.) (1983) Meth. Enzymol. 100 & 101;Grossman and Moldave (eds.) (1980) Meth. Enzymol. 65; Miller (ed) (1972)Exp. Mol. Genetics, Cold Spring Harbor, N.Y.; Old and Primrose (1981)Principles of Gene Manipulation, Univ. of Cal. Press, Berkely; Schliefand Wensink (1982) Practical Methods in Molecular Biology; Glover (ed)1985(DNA Cloning, Vols. I and II, IRL Press, Oxford, UK; Sellow andHollaender (1979) Genetic Engineering: Principles and Methods, Vols I,Plenum Press, NY; which are incorporated by reference in their entiretyherein. Abbreviations, where employed, are those deemed standard in thefield and commonly used in professional journals such as those citedherein.

Expression of a gene requires both transcription of DNA into mRNA andthe subsequent translation of the mRNA into a protein. Because generegulation usually occurs at the level of transcription, transcriptionalregulation and activity of GDF promoters of the present invention can beassayed by quantitation of gene products. For example, promoterregulation and activity can be quantitatively studied by the fusion ofthe easily assayable E. Coli lacZ gene sequence to a heterologouspromoter (Casadaban and Cohen (1980) J. Mol. Biol. 138:179-207).Alternatively, the genes coding for chloramphenicol acetyl transferase(CAT), green fluorescence protein (GFP) and luciferase can be used todetect activity of a promoter. Such genes are termed “reporter” geneswhich, when combined with a given promoter (usually a heterologouspromoter), provide a ready assay for promoter activity.

GDF promoters and GDF promoter elements of the invention (i.e., selectedsequences within GDF-8 promoters involved in their regulatory function)may be employed in the form of single or multiple units, in numerousvarious combinations and organizations, in forward or reverseorientations, and the like. In the context of multiple unit embodimentsand/or in embodiments which incorporate both positive and negativecontrol elements, there is no requirement that such units be arranged inan adjacent head-to-head or head-to-tail construction since the improvedregulation capability of such multiple units is conferred virtuallyindependent of the location of such multiple sequences with respect toeach other. Moreover, there is not requirement that each unit comprisethe same positive or negative element. Such sequences can be locatedupstream of and sufficiently proximal to a transcription initiationsite, in the intron or downstream of the gene of interest, to confer adesired regulatory effect. In addition, GDF promoter and GDF promoterelements of the invention can be used in numerous various combinationswith promoters and regulatory elements of other genes to achieve thedesired enhancement or repression of the expression of any gene ofinterest.

Accordingly, in one embodiment of the invention, the GDF promoter isused to regulate transcription of a heterologous structural gene bysimply obtaining the structural gene and inserting one or more copies ofthe GDF promoter upstream of the gene's transcription initiation site.Additionally, as is known in the art, it is generally desirable toinclude TATA-box sequences upstream of and proximal to the transcriptioninitiation site of the heterologous structural gene. Such sequences maybe synthesized and inserted in the same manner as the novel controlsequences of the invention. Alternatively, the TATA sequences naturallyassociated with the heterologous structural gene can be employed.Generally, the TATA sequences are located between about 20 and 30nucleotides upstream of transcription initiation.

Numerous methods are known in the art for precisely inserting selectednucleotide sequences, at selected points, within larger sequences. Inone method, the desired control sequences, or combinations of sequences,are synthesized and restriction site linker fragments added to thecontrol sequence termini. This allows for ready insertion of the controlsequences into compatible restriction sites within upstream regions.Alternatively, synthesized control sequences can be ligated directly toselected regions. In addition, site specific mutagenesis can be employedto fashion restriction sites into which control sequences may beinserted, in the case where no convenient restriction sizes are found ata desired insertion site.

GDF promoters of the present invention can be beneficially employed inthe context of any heterologous gene, with or without additionalhomologous or heterologous control or promotion sequences. In aparticular embodiment, the present invention provides the GDF-8 genepromoter and optionally other regulatory sequences which function in theinduction of GDF-8 expression in response to factors which are known toinduce GDF-8 expression.

Any suitable reporter gene can be used to measure the activity of a GDFpromoter element of the invention. For example, in the examplesdescribed below, a GDF-8 promoter-luciferase reporter construct wasused. The GDF-8 promoter-luciferase reporter construct was active in twocell lines (FIGS. 1A-1B), RD (human embryonal rhabdomyosarcoma) and A673(human rhabdomyosarcoma). Maximum activity was observed with the 0.65 Kb5′ flanking fragment (5-8% of the SV40 luciferase reporter plasmid,pGL3-control). No significant luciferase activities were detected in theHepG2 (human liver hepatoblastoma), HT29 (human colon adenocarcinoma),PA-1 (human ovarian teratorcarcinoma), and MCP-7 (human breastadenocarcinoma). The results of the transfection assays suggest that 1)a repressor element(s) exists between 8.5 Kb and 0.65 Kb upstream of thehuman GDF-8 gene, 2) the 0.65 Kb fragment contains the minimal promoter,and 3) the minimal promoter is tissue specific and appears to be activein muscle cell lines only.

In yet another embodiment, the present invention provides a method ofscreening for a compound which binds to a GDF promoter (or a portionthereof), such as a GDF-8 promoter), and modulates expression of a GDFgene (e.g., GDF-8) or a heterologous gene. As used herein, the term“modulate” includes both inhibition and stimulation of GDF expression.Moreover, the term “inhibition” is intended to include both complete andpartial inhibition of GDF expression. In various embodiments, GDFexpression is inhibited to a level at least 1.2-fold, 1.5-fold,1.8-fold, 2-fold, 2.5-fold, 3-fold, 4-fold or 5-fold lower than the wildtype level of GDF-8 expression. In further embodiments, GDF expressionis inhibited by at least 10%, 20%, 30%, 40%, 50%, 75% or 100%.

To test compounds for their ability to modulate GDF transcription and/orexpression, the compound can be tested for its ability to increase orstimulate the transcription and/or expression or to decrease or inhibitthe transcription and/or expression of a reporter gene (e.g., the SV40β-galactosidase reporter gene) which is operatively linked to a GDFpromoter of the invention, compared to that of a control reporter.Effects of the test compound are determined by changes in reporter geneactivity. For example, a stable cell line containing a GDF-8 promoteroperatively linked to a reporter gene, such as the luciferase gene, canbe used in any well-known screening method known in the art fordetecting expression (e.g., luciferase assays, CAT assays, or GreenFluorescent Protein (GFP) assays). However, the invention is notrestricted to these suggested screening methods.

Transcription factors that bind to GDF promoters of the invention alsocan be characterized using gel mobility shift assays and thesetranscription factors can be cloned using these specific sequences asprobes in screening expression libraries. Alternatively, secondgeneration reporter constructs containing multiple copies of thefollowing transcription factor binding sequences: CAAATG, CAGACA orGACAGC and a minimal promoter (0.2 kb upstream of initiating ATG), or acombination of these sequences and a minimal promoter, can be used inhigh-throughput screening assays to identify inhibitors specific for GDFgenes which operate by binding to GDF promoter sequences. After thesetranscription factors have been identified, they too, may be used astargets for identifying other inhibitors. For example, thesetranscription factors can be used as “bait proteins” in a two-hybridassay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervoset al. (1993) Cell 72:223-232; Madura et al. (1993) J Biol. Chem.268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchiet al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), or as proteinprobes in the screening of expression libraries to identify otherproteins, which bind to or interact with the transcription factors. Suchtranscription factor-binding proteins are also likely to act asmodulators, e.g., inhibitors of GDF-8 expression.

In one embodiment, the invention provides a GDF-8 promoter, or a portionthereof, which can be operatively linked to a gene of interest (GOI) andexpressed in a tissue-specific manner. GDF-8 promoter activity isspecific to muscle tissue. Therefore, the GDF-8 promoter can be used toexpress, any GOI for which expression is desired in muscle tissue.Examples of such genes include, but are not limited to, GDF-8 itself,dystrophin, growth factors, genes coding for tumor or pathogensantigens. Genes which express proteins useful in vaccination are alsoencompassed, including viral, tumor, pathogenic, or bacterial antigens,specifically AIDS envelope protein gp120. However, this is not intendedto be a limiting list. Any gene which expresses a protein of interestmay be employed in the methods of the invention.

Expressing a protein of interest specifically in muscle tissue is highlypreferred in the area of gene therapy due to the amount of muscle massin the body and the ease in which muscle can express foreign genes.Accordingly, the GDF-8 promoter of the invention can be used in genetherapy vectors to direct expression of a gene of interest in muscletissue. Gene therapy vectors including the GDF-8 promoter of theinvention can be delivered to a subject by, for example, intravenousinjection, local administration (see U.S. Pat. No. 5,328,470),stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad.Sci. USA 91:3054-3057), or direct intramuscular injection (as describedin, for example, U.S. Pat. Nos. 5,580,859 and 5,589,466). The genetherapy vectors containing the GDF-8 promoter of the invention can beused for the treatment of a muscle-associated disorder such as cancer,muscular dystrophy, spinal cord injury, traumatic injury, congestiveobstructive pulmonary disease, AIDS or cachexia; or for the treatment ofobesity and related disorders, e.g., diabetes; or disorders related toabnormal proliferation of adipocytes.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are incorporated herein by reference.

EXAMPLES Example 1

Screening of Human Genomic Library

A Stratagene human genomic library (HT1080) was screened using a 745 bpEcoRI-Hind III human GDF-8 cDNA probe (described in, for example, U.S.patent application Ser. No. 08/525,596). Three genomic clones wereisolated using methods described in Current Protocol in MolecularBiology, Eds. Ausubel et al., John Wiley & Sons, Inc., 996). An 8.5kilobase (Kb) fragment containing the 5′ flanking region of human GDF-8was subcloned into the luciferase reporter construct pGL-3 -basic(Promega). This 8.5 kilobase fragment was truncated by a restrictiondigestion or by PCR to generate six additional clones: 5.3 Kb, 3.6 Kb,0.65 Kb, 0.3 Kb, 0.2 Kb, and 0.1 Kb. Approximately 1 Kb upstream, of theinitiating ATG has been sequenced. The approximate transcription startsite, based on the positions of the CCAAT and TATAA boxes is about 100bases upstream of the ATG. Therefore, the luciferase clone containing0.1 Kb contains only the 5′ untranslated region.

Example 2

Transfection Assays

Transient transfection assays were performed using FuGENE (BoehringerMannheim) according to the manufacturer's protocol. Six different humancell lines, RD, A673, HepG2, HT29, PA-1, and MCF-7 (ATCC, Rockville,Md.) were used in the transfection assays. On the day beforetransfection, 1.5×10⁵ cells in 2 ml of DMEM with 10% FBS were seeded in35 mm tissue culture dishes. The cells were incubated overnight untilthe cells were 50-70% confluent. For each transfection, 1.0 μg ofluciferase reporter plasmid, 0.1 μg of β-galactosidase reporter plasmid,pSV-β-gal (Promega), and 5 μg of FuGENE was used. A promoter-lessluciferase vector (pGL3-basic)(Promega) and a SV40 luciferase vector(pGL3-control)(Promega) were used as controls. Luciferase andβ-galactosidase activities were determined 24 hours post-transfectionusing the Dual Light chemiluminescent reporter gene assay kit (Tropix,Inc.) according to the manufacturer's protocol. The relative activitiesof the luciferase reporter constructs were normalized to theβ-galactosidase activity.

Example 3

GDF-8 Promoter Mutants

Additional truncations of the GDF-8 promoter were made using PCR togenerate DNA fragments containing 0.65 Kb, 0.44 Kb, 0.31 Kb, 0.29 Kb,0.25 Kb, 0.21 Kb, and 0.1 Kb of sequence upstream of the initiating ATGcodon (see FIGS. 2 and 3A). These DNA fragments were then subeloned intothe luciferase reporter plasmid, pGL3-basic (Promega). The constructs(0.65 Kb to 0.1 Kb) were transfected into RD cells and the expression ofthese constructs was determined using luciferase assays (see FIGS. 3Aand 3B). Deleting the region from 0.31 Kb to 0.29 Kb decreased theluciferase activity by about 40%; from 0.31 Kb to 0.25 Kb decreased theluciferase activity by about 60%; and from 0.31 Kb to 0.21 Kb decreasedthe luciferase activity by about 90%. Examination of the regions from0.31 Kb to 0.29 Kb, and 0.29 Kb to 0.25 Kb, revealed the sequence CAAATG(potential E-box) and CAGACA (potential Smad 3 and 4 binding sequence),respectively. Although examination of the 0.25 Kb to 0.21 Kb region didnot reveal any known transcription binding sites, the deletion resultssuggest the possibility of a cis-element.

To determine whether the decrease in luciferase activity from thedeletion constructs was due to the regulatory role of the CAAATG, CAGACAand GACAGC sequences in GDF-8 expression, or due to the physicaltruncation of the promoter region, clustered site-directed mutagenesis,using the QuikChange™ site-directed mutagenesis kit (Stratagene), wasperformed. The following mutants were created (CAAATG→AGATCT;CAGACA→AGATCT; and GACAGC→AGATCT). In addition, GDF-8 promoter reporterconstructs containing multiple clustered mutations were also generated.The mutated promoter constructs were transfected into RD cells andassayed for luciferase activity (see FIGS. 4A and 4B). Mutating theCAAATG region resulted in approximately 25% decrease in luciferaseactivity; mutating the CAGACA region resulted in an increase inluciferase activity; and mutating the GACAGC region resulted inapproximately 40% decrease in luciferase activity.

These results suggest that not only does the physical truncation of thepromoter region have an effect on luciferase expression, but moreimportantly, that the sequences CAAATG, CAGACA and GACAGC play aregulatory role in expression. The two sequences, CAAATG and GACAGC,also appear to act synergistically as demonstrated by a 60% decrease inluciferase activity in the double mutant. These results also suggestthat there are multiple regulatory regions (e.g., transcription factorbinding regions) in the GDF-8 promoter. Therefore, GDF-8promoter-luciferase gene reporter constructs can be used inhigh-throughput screenings (HTS) for inhibitors of GDF-8 expression mayyield inhibitors acting at different regulatory sites.

Example 4

Generation of Expression Constructs Suitable for Use in Screening forRegulatory Compounds of GDF-8 Expression

The mutation of the sequences CAAATG, CAGACA and GACAGC results ineither inhibiting (for CAAATG and GACAGC) or enhancing (for CAGACA) theexpression of the GDF-8 promoter construct pGL3-0.65. This indicatesthat these sequences are involved in the regulation of GDF-8 expressionand suggests that these sequences are recognized by transcriptionfactors. To demonstrate this, Luciferase expression plasmids containinga minimal promoter sequence (pGL3-0.21 containing the region −1 to −207,FIG. 2) and one or more copies of the above regulatory sequencesupstream of the minimal promoter sequence were constructed. FIG. 5Ashows the double stranded oligonucleotide sequences containing thesequences CAAATG and GACAGC and their flanking sequences used in thegeneration of concatemers (multiple copies of the above-identifiedsequences). Expression of the luciferase reporter constructs containingthe CAAATG sequence was dependent on the number of copies of thissequence contained within the construct, while the expression of theluciferase reporter constructs remains 100% of the control regardless ofthe number of copies of the GACAGC sequence (FIG. 5B). Thesetransfection results indicate that these expression plasmids can be usedin screening protocols to identify compounds that regulate specifictranscription factors interacting with the above regulatory sequences.

The approaches described in this Example, as well as Examples 1, 2 and 3above, can also be used to identify any other unknown regulatorysequences in the human GDF-8 promoter and other GDF-8 promoter,including but not limited to mouse, pig or chicken GDF-8 promoters. Inaddition, the various expression constructs described in this Example,as well as Examples 1, 2 and 3 above, can be used to generate transgenicanimals to demonstrate promoter and/or regulatory activity in vivo.

Example 5

Generation of Cell Lines Suitable for Use in Screening for RegulatoryCompounds of GDF-8 Expression

Stable cell lines containing the pGL3-0.65 plasmid sequence weregenerated by transfecting RD cells with the pGL3-0.65 plasmid andselecting for stable cell clones under G418 pressure selection. Theexpression profiles of two such stable cell clones are shown in FIG. 6.Treatment of these cell clones with TGF-β or TNFα down regulated theluciferase reporter gene expression demonstrating that the exogenousGDF-8 promoter can be regulated and that these cell clones are useful inthe screening and identification of GDF-8 expression regulatorycompounds. Stable cell lines containing CAAATG and GACAGC concatemerreporter plasmids (described in Example 4) can also be generated for usein the screening and identification of GDF-8 expression regulatorycompounds, e.g., compounds that specifically affect regulatory proteins(such as transcription factors) binding to these sequences.

Example 6

Regulatory Sequences in GDF-8 Promoters Are Conserved Among VariousSpecies

Additional promoter sequences from chicken and mouse GDF-8 were obtainedby sequencing a chicken GDF-8 genomic clone, isolated by screening aWhite Leghorn Chicken genomic library (Stratagene), and by screening amouse GDF-8 genomic clone, kindly provided by Dr. Se-jin Lee at JohnsHopkins University (see McPherron et al. (1997) Nature 387:83-90). Thenucleotide sequence for the pig GDF-8 gene can be obtained from GenBankAccession numbers AJ133580 and AF093798.

A comparison of human, mouse, chicken and pig GDF-8 promoter sequences(160 nucleotides upstream of the TATAA box) is shown in FIG. 8 andreveals a high level of sequence homology between the four species (seeTable 1). In particular, the regulatory sequences CAAATG and GACAGC arepresent within this region in all four species. The high degree ofsequence identity in the promoter regions of these species and theconservation of these regulatory sequences in this region indicates thatthe same transcription factors that bind to the human promoter also canrecognize regulatory sequences in pig, mouse and chicken. Therefore, thesame strategies that can be used to identify regulatory sequences in thehuman promoter can also be used to identify regulatory sequences in thepromoter regions of mouse, pig and chicken GDF-8.

The activity of the promoter and regulatory elements of the mouse, pigand chicken GDF-8 genes can be determined and analyzed using methodsdescribed for the human GDF-8 gene in Examples 1, 2, 3 and 4. Forexample, cells and cell lines of mouse, pig and chicken origin can beused in the in vivo analysis of mouse, pig and chicken GDF-8 promoterand regulatory elements, respectively. Since the GDF-8 promoter regionis highly conserved among the different species, in addition to usingcells and cell lines from homologous species in analyzing promoter andregulatory element activities in in vivo assays, cells and cell lines ofheterologous species may also be used. Additionally, the GDF-8 promoterand regulatory element activities of any species including, but notlimited to, human, mouse, pig and chicken, can be analyzed in vivo bythe generation of transgenic animals using reporter vectors containingthe GDF-8 promoter, portion of the promoter, regulatory elements and/orcombinations thereof. TABLE 1 % Sequence Homology Human Pig MouseChicken Human 100 97 96 79 Pig 100 95 78 Mouse 100 76 Chicken 100Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method of identifying a compound which regulates GDF-8 expressioncomprising operably linking to a gene an isolated GDF-8 promotercomprising the nucleotide sequence of SEQ ID NO:1, contacting thepromoter with the compound in a cultured cell such that the gene istranscribed and expressed, and measuring expression of the gene comparedto a control where the compound is absent
 2. The method of claim 1further comprising the step of comparing expression of the gene aftercontacting the promoter with the compound to expression of the genewithout contacting the promoter with the compound, to determine whetherthe compound has effected expression of the gene.
 3. The method of claim1, wherein the gene is a reporter gene.
 4. The method of claim 1,wherein the compound inhibits GDF-8 expression.
 5. The method of claim1, wherein the compound upregulates GDF-8 expression.
 6. A method ofidentifying a compound which regulates GDF-8 expression comprisingoperably linking to a gene an isolated GDF-8 promoter comprising anucleotide sequence which is at least 95% identical to the entirenucleotide sequence of SEQ ID NO:1, contacting the promoter with thecompound in a cultured cell such that the gene is transcribed andexpressed, and measuring expression of the gene compared to a controlwhere the compound is absent.
 7. The method of claim 6 furthercomprising the step of comparing expression of the gene after contactingthe promoter with the compound to expression of the gene withoutcontacting the promoter with the compound, to determine whether thecompound has effected expression of the gene.
 8. The method of claim 6,wherein the gene is a reporter gene.
 9. The method of claim 6, whereinthe compound inhibits GDF-8 expression.
 10. The method of claim 6,wherein the compound upregulates GDF-8 expression.
 11. A method ofidentifying a compound which regulates GDF-8 expression comprisingoperably linking to a gene an isolated GDF-8 promoter comprising anucleotide sequence which is at least 90% identical to the entirenucleotide sequence of SEQ ID NO:1, contacting the promoter with thecompound in a cultured cell such that the gene is transcribed andexpressed, and measuring expression of the gene compared to a controlwhere the compound is absent.
 12. The method of claim 11 furthercomprising the step of comparing expression of the gene after contactingthe promoter with the compound to expression of the gene withoutcontacting the promoter with the compound, to determine whether thecompound has effected expression of the gene.
 13. The method of claim11, wherein the gene is a reporter gene.
 14. The method of claim 11,wherein the compound inhibits GDF-8 expression.
 15. The method of claim11, wherein the compound upregulates GDF-8 expression.